Method of detection and quantification of cGMP by using a cGMP-visualizing probe

ABSTRACT

As a cGMP-visualizing probe capable of detecting and quantifying cGMP easily and accurately even in vivo and for a method of detecting and quantifying cGMP by using the same, a cGMP-visualizing probe comprising a polypeptide, which binds specifically to cGMP, and two chromophores having different fluorescence wavelengths each linked respectively to the two terminals of said polypeptide is provided.

This application is a divisional of Ser. No. 10/070,131, filed Apr. 1,2002, now U.S. Pat. No. 6,924,119, which is a 371 U.S. National Stage ofPCT/JP01/05631, filed Jun. 29, 2001.

TECHNICAL FIELD

The invention of the present application relates to a visualizing probefor detecting and quantifying cGMP and a method of detecting andquantifying cGMP by using the same. More specifically, the presentinvention relates to a cGMP-visualizing probe which can specificallybind to cGMP, thereby generating an optical change to enable detectionand quantification of cGMP, and a method of detecting and quantifyingcGMP by using the same.

BACKGROUND ART

Cyclic guanosine monophosphate (cGMP) is an intracellular secondmessenger which acts as a signal molecule in various biochemicalreaction processes in living body. So far it has been revealed thatvarious physiological processes such as relaxation of vascular musclecells, photo transduction in retina, epithelial electrolyte transport,bone growth, and neuronal activity are regulated by cGMP. Accordingly,if the synthesis, decomposition and localization of cGMP in living cellscan be clarified, not only would the mechanism of cGMP at the cellularand tissue levels in the circulatory system, in the kidney and retina,in the olfactory and central nerves be understood, but would alsoprovide hints for obtaining knowlege on substances such asphosphodiesterase inhibitors, which control intracellular cGMP levels inthe cell.

Conventional methods of detecting and quantifying cGMP include radioimmuno assays using a radioisotope-labeled compound. However, thismethod involves disrupting cells and detecting the binding of cellularlysates to the labeled compound to analyze total cGMP levels, and didnot realize the accuracy required in cell-biology and pharmaceutics.

Further, since such radioimmunoassay is a destructive measurementmethod, and further because the radioisotope-labeled compound is poor instability and requires caution in handling, such conventional methodcould only be applied only to in vitro measurement of cGMP; in thisrespect, there was a limit to which the method could be applied.

The invention of the present application has been made in view of thecircumstances as described above, and the object of the presentinvention is to solve the problem in the prior art and to provide acGMP-visualizing probe, which enables the easy and highly accuratedetection and quantification of cGMP, even in vivo, as well as a methodof detecting and quantifying cGMP by using the same.

DISCLOSURE OF INVENTION

To solve the problem described above, the invention of the presentapplication first provides a cGMP-visualizing probe, comprising apolypeptide that binds specifically to cGMP and two chromophores withdifferent fluorescence wavelengths, which are each linked to the twoterminals of the polypeptide.

Secondly, the invention of the present application provides thecGMP-visualizing probe, wherein the polypeptide that binds specificallyto cGMP is a cGMP-binding protein.

Thirdly, the invention of the present application provides thecGMP-visualizing probe, wherein the polypeptide that binds specificallyto cGMP is cGMP-dependent kinase Iα.

Fourthly, the invention of the present application provides thecGMP-visualizing probe, wherein the chromophores are cyan fluorescentprotein linked to the N-terminal of the polypeptide and yellowfluorescent protein linked to the C-terminal of the polypeptide.

Fifthly, the invention of the present application provides a method fordetecting and quantifying cGMP, which comprises making thecGMP-visualizing probe coexist with-cGMP; and measuring the change inthe fluorescence wavelength.

Sixthly, the invention of the present application provides the methodfor detecting and quantifying cGMP, which comprises introducing apolynucleotide expressing the cGMP-visualizing probe into cells, therebymaking the cGMP-visualizing probe coexist with cGMP.

Seventhly, the invention of the present application provides the methodfor detecting and quantifying cGMP, which comprises introducing apolynucleotide expressing a cGMP-visualizing probe into cells andperforming ontogenesis from the non-human animal totipotent cells,thereby making the cGMP-visualizing probe coexist with cGMP in everycell of the resultant animal or its offspring.

Eighthly, the invention of the present application provides a non-humananimal or offspring thereof, which is obtained by introducing apolynucleotide expressing a cGMP-visualizing probe into cells andperforming ontogenisis from the non-human animal totipotent cells.

Ninthly, the invention of the present application provides a method forscreening a substance, which comprises introducing a test samplecontaining the substance into a non-human animal or offspring thereof,and quantifying cGMP in the cells of the non-human animal or offspringthereof.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic representations of the structures of thecGMP-visualizing probe of the present invention.

FIG. 2 shows the response to 8-Br-cGMP in CHO-K1 cells expressing thecGMP-visualizing probes for the Examples of the present invention. (a)is a microphotograph of the cells expressing CGY-FL, CGY-A12 andCGY-Del1 (excitation: 440±10 nm, filter: 480±15 nm (CFP) and 535±12.5 nm(YFP)). (b) shows the change in emission ratios at 480±15 nm to 535±12.5nm with time for the CGY-expressing CHO-K1 cells coexisting with 1 mM8-Br-cGMP.

FIG. 3 is a graph showing the change with time in emission ratios uponstimulation of CGY-FL-, CGY-Del1- and CGY-A12-expressing CHO-K1 cellswith 8-Br-cGMP for the Examples of the present invention. (a) is forCGY-FL, (b) for CGY-Del1, and (c) for CGY-A12 respectively.

FIG. 4 is a graph showing the kinetic behavior of the cytoplasmicfluorescence intensity of CGY-Del1-expressing CHO-K1 cells at 480±15 nm(CFP) and 535±12.5 nm (YFP) for the Examples of the present invention.(a) is for CFP, and (b) for YFP respectively.

FIG. 5 is a graph showing initial emission ratios and changes in theemission ratios for CGY-FL, CGY-A12, CGY-Del1, CGY-Del2, CGY-Del3, andCGY-Del4 in the Examples of this invention.

FIG. 6 shows the cytoplasmic emission ratios upon stimulation ofCGY-Del1-expressing HEK293 cells with 500 μM of NOC-7 for the Examplesof the present invention (-Br-cGMP added 900 seconds after addition ofNOC-7 8.)

FIG. 7 is a graph showing the changes in emission ratios uponstimulation of CGY-Del1-expressing HEK293 cells with 500 μM NOC-7 forthe Examples of the present invention. (a) shows CGY-Del1-expressingHEK293 cells, (b) shows CGY-Del1-expressing HEK293 cells treated with100 μM zaprinast, and (c) CGY-Del1-expressing HEK293 cells treated with10 μM ODQ.

FIG. 8 is a graph showing changes in the emission ratios uponstimulation of CGY-Del1-expressing HEK293 cells with 500 μM of NOC-7 inthe Examples of this invention. (a) 8-Br-cGMP added 900 seconds afteraddition of NOC-7; (b) zaprinast added 900 seconds after addition ofNOC-7.

BEST MODE FOR CARRYING OUT THE INVENTION

The cGMP-visualizing probe of the invention of the present applicationcomprises two sites each having a different function. That is, thiscGMP-visualizing probe comprises a site that selectively recognizes cGMPand binds specifically to cGMP, and coloring sites that transmit opticalsignals upon recognition of cGMP by the site that binds specifically tocGMP.

The detection and quantifcation of cGMP is enabled when thecGMP-visualizing probe of this invention coexists with cGMP; the sitethat binds specifically to cGMP (cGMP-binding site) binds to cGMP,causing the change of configuration of the coloring sites, which may bedetected as an optical change.

The site for binding specifically to cGMP is, for example, a polypeptidesuch as various cGMP-binding proteins. The cGMP-binding proteins includecGMP-dependent protein kinase Iα (PKG Iα).

Mammalian PKG Iα is composed of two identical monomers each having fourtypes of functional domains (dimerization domain, autoinhibitory domain,cGMP-binding domain and catalytic domain) as shown in FIG. 1. Thedimerization domain located at the N-terminus is composed of aleucine/isoleucine zipper motif. In the absence of cGMP, PKG Iα displaysa kinase inactive closed conformation, in which its catalytic center isoccupied by an autoinhibitory domain. Upon binding of PKG Iα to cGMP,PKG Iα displays an open conformation in which the autoinhibitory domainis removed from the catalytic center. Accordingly, if chromophores arelinked to both terminals of PKG Iα, an optical change will occur uponbinding of PKG Iα to cGMP, thus allowing the optical detection of thebinding to cGMP.

As a matter of course, the polypeptide binding specifically to cGMP isnot limited to PKG Iα or cGMP-binding proteins, and every possiblesynthetic or natural peptide chain may be used.

In the cGMP-visualizing probe of the invention of the presentapplication, the site transducing the molecular recognition to anoptical change may be chosen from various chromophores. In thistransduction, the chromophores should generate a change in wavelengthhighly accurately by responding to the change in the stereostructureresulting from the binding of the cGMP-binding site to cGMP. In thefield of biochemistry, there are a wide variety of generally usedfluorescent chromophores including, as chromophores responding rapidlyto a change in the stereostructure, those causing change in color by thegeneration of fluorescence resonance energy transfer (FRET).

As the sites transducing the molecular recognition event to an opticalchange in the cGMP-visualizing probe of the invention of the presentapplication, two fluorescent chromophores each having a differentfluorescence wavelength are linked respectively to the two terminals ofthe polypeptide binding specifically to cGMP. As such fluorescentchromophores, cyan fluorescent protein (CFP) i.e. a blue-shifted mutantof green fluorescent protein (GFP) and yellow fluorescent protein (YFP)i.e. a red-shifted mutant of GFP are preferably selected. By linking CFPto the N-terminal of the polypeptide binding specifically to CGMP andYFP to the C-terminal thereof, the two act respectively as donor andacceptor to generate FRET.

That is, when the cGMP-visualizing probe of the invention of the presentapplication coexists with cGMP, the cGMP-binding protein binds to cGMPto allow FRET to be generated by the fluorescent chromophores at the N-and C-terminals thereof, thus causing a change in fluorescencewavelength. Then, cGMP may be detected by measuring such fluorescencechange by a variety of conventional chemical and/or biochemical analysistechniques. Further, the concentration of cGMP in a sample solution mayalso be quantified by previously calibrating the relationship betweenfluorescence intensity and cGMP concentration.

In the invention of the present application, various methods areapplicable for the cGMP-visualizing probe described above to coexistwith cGMP. For example, a method wherein cells are disrupted, cGMP iseluted from the cells, and the cGMP-visualizing probe is added to thesolution, to allow the cGMP-visualizing probe to be coexistent with cGMPmay be applied. When the cGMP-visualizing probe is allowed to becoexistent with cGMP by such a method, cGMP can be detected andquantified in vitro.

In the present invention, by introducing an expression vector having thecGMP-visualizing probe integrated therein into individual culturedcells, the cGMP-visualizing probe may also be made to coexist with cGMP.For such a method, the expression vector, a plasmid vector forexpression in animal cells is preferable. The introduction of suchplasmid vectors into cells, may be accomplished by known methods such aselectroporation, calcium phosphate method, liposome method andDEAE-dextran method. By using the method of introducing an expressionvector having the cGMP-visualizing probe integrated therein into cells,cGMP and the cGMP-visualizing probe may coexist in the cells, thusenabling an in vivo method of detecting and quantifying cGMP withoutdisrupting the cells.

Further, in the method of detecting and measuring cGMP by the inventionof the present application, a polynucleotide expressing thecGMP-visualizing probe may be introduced into cells, and by ontogenesis,the non-human animal totipotent cells may be generated into anindividual non-human animal, for the cGMP-visualizing probe to coexistwith cGMP in every cell of the resultant animal or its offspring.

Using the various methods described above, a transgenic non-human animalhaving the cGMP-visualizing probe and cGMP coexisting in every cellthereof may be obtained in the invention of the present application byintroducing a polynucleotide expressing the cGMP-visualizing probe intocells and developing the non-human animal totipotent cells into anindividual. The transgenic non-human animal may be created by knownpreparative methods (for example, Proc. Natl. Acad. Sci. USA77:7380-7384, 1980). The transgenic non-human animal obtained would havethe cGMP-visualizing probe in every somatic cell, and may be used, forexample, in the screening of various substances by introducing testsubstances such as pharmaceutical preparations or toxins to the body ofsaid transgenic animal and measuring the concentration of cGMP in thecells and tissues.

Hereinafter, the present invention is described in more detail byreference to the Examples and the accompanying drawings. As a matter ofcourse, the invention is not limited to the following examples, andvarious embodiments are possible.

The cGMP-visualizing probe of the invention of the present applicationis genetically encoded; thus it may be optimized by genetic engineeringmeans. For example, the position of the cGMP-visualizing probe in thecell may be controlled by fusing it to a protein such as a signalsequence, guanylyl cyclase, a cyclic nucleotide-agonistic cationchannel, and an anchoring protein for PKG Iα.

Further, similar to the cGMP-visualizing probe of the invention of thepresent application, cyclic adenosine-phosphate (cAMP) visualizingprobes may be developed based on knowledge on genetic engineering inrelation to the binding domains of cyclic nucleotides.

EXAMPLES Example 1 Preparation of cGMP-Visualizing Probes

As shown in FIG. 1, cyan fluorescent protein (ECFP:F64L/S65T/Y66W/N1461/M153T/V163A/N212K) and yellow fluorescent protein(EYFP: S65G/V68L/Q69K/S72A/T203Y), which are mutants of greenfluorescent protein (EGFP: for example, Current Biology 6(2), 178-182,1996) derived from fluorescent Aequorea victoria, were linkedrespectively to the N- and C-terminals of cGMP-dependent protein kinase(PKG Iα) by genetic engineering, to prepare a CFP-PKG Iα-YFP fusionprotein (referred to hereinafter as CGY).

Besides this CGY construct (CGY-FL) having the full-length amino acidsequence of PKG Iα, the following CGY constructs were prepared in ananalogous method: a CGY construct (CGY-A12) wherein in the amino acids 1to 47 in PKG Iα, all of leucine, isoleucine and cysteine residues wereconverted into alanine residues, and a CGY construct (CGY-Del1) that isPKG Iα (Δ1-47) wherein the amino acids 1 to 47 in PKG Iα were deleted.

Further, the following an alogues of CGY-Del1 were prepared in ananalogous manner: a construct (CGY-Del2) wherein the linker sequence(MDELKY) of SEQ ID NO: 1 was introduced between PKG Iα (Δ1-47) and ECFP,a construct (CGY-Del3) wherein the linker sequence (YPYDVPDYAN) of SEQID NO: 2 was introduced between PKG Iα (Δ1-47) and EYFP, and a construct(CGY-Del4) wherein the linker sequence (MDELKY) of SEQ ID NO: 1 wasintroduced between PKG Iα (Δ1-47) and ECFP and the linker sequence(YPYDVPDYAN) of SEQ ID NO: 2 was introduced between PKG Iα (Δ1-47) andEYFP.

Example 2 Introduction of the cGMP-Visualizing Probes (CGY) into ChineseHamster Ovary Cells

Chinese hamster ovary cells (CHO-K1) were cultured in Ham's F-12 mediumsupplemented with 10% fetal calf serum (FCS) at 37° C. in 5% CO₂.

The resulting CHO-K1cells were transfected by Lipofect AMINE 2000reagent (Life Technologies) with CGY-FL, CGY-Del1 and CGY-A12 expressionvectors respectively.

Within 12 to 24 hours after the transfection, the CHO-K1 cellsexpressing each CGY were removed and spread onto glass-bottom culturedishes.

Example 3 Imaging of CHO-K1 into which the cGMP-Visualizing Probes (CGY)had been Introduced

First, the culture liquid in Example 2 was replaced with Hank's balancedsalt solution.

Within 3 to 5 days after transfection with each type of CGY, the CHO-K1cells were imaged at room temperature on a Carl Zeiss Axiovert 135microscope with a cooled CCD camera MicroMAX (Roper Scientific Inc.)controlled by MetaFluor (Universal Imaging). The exposure time at 440±10nm excitation was 50 ms. The fluorescence images were obtained through480±15 nm and 535±12.5 nm filters with a 40×oil-immersion objective(Carl Zeiss).

FIG. 2( a) shows a microphotograph of CFP (480 nm) and YFP (535 nm) ofthe transfected cells. From this result, it was revealed that CGY-A12was introduced into the cell nucleus.

Since about 30% of CHO-KL cells expressing CGY-Del1 showed CFP and YFPfluorescence in the cell nucleus, the leucine/isoleucine zipper motif inthe dimerization domain of PKG Iα may provide important information tothe direct localization of kinase in the extranuclear compartment.

Example 4 Responses of the cGMP-Visualizing Probes (CGY)

The CGY-expressing CHO-K1 cells prepared in Example 1 were stimulatedwith 8-Br-cGMP known as a cell membrane-permeable andphosphodiesterase-resistant-analogue of cGMP, and the fluorescence wasmeasured under a fluorescence microscope.

(1) CGY-FL-expressing CHO-K1

FIG. 2( b) shows changes in emission ratios (480±15 nm to 535±12.5 nm)in the CGY-expressing CHO-K1 before and after the addition of 8-Br-cGMP(1 mM).

Further, FIG. 3 shows changes with time in emission ratios in the cellsubstrate.

From these results, it was revealed that there is no significantemission ratio change of CGY-FL. It was also revealed that thefluorescence intensities of CFP and YFP were not affected by theaddition of 8-Br-cGMP.

(2) CGY-Del1-Expressing CHO-K1

On the other hand, a significant decrease in the emission ratio for theCGY-Del1-expressing CHO-K1 upon addition of 8-Br-cGMP was confirmed asshown in FIG. 2 b.

Further, as shown in FIG. 4, reciprocal changes in CFP and YFPfluorescence intensities were observed. Further, it was found that thedecrease in the emission ratios has the same tendency with time as forthe reciprocal changes in CFP and YFP fluorescence intensities.

These results indicate that the fluorescence resonance energy transfer(FRET) between CFP and YFP increases upon binding of 8-BR-cGMP toCGY-Del1.

(3) CGY-A12-Expressing CHO-K1

In CGY-A12-expressing CHO-K1 cells, a change in the emission wasobserved by the addition of 8-Br-cGMP (FIGS. 2 b, 3). However, themaximum change in this emission ratio was as small as one-third of thatfor CGY-Del1.

Example 5 Influence of Introduction of the Linker Sequences into thecGMP-Visualizing Probes

CGY-Del1, CGY-Del2, CGY-Del3 and CGY-Del4 prepared in Example 1 wereexpressed in CHO-K1 cells in the same manner as in Example 2, andexamined for changes in the emission intensities.

The relationship between the initial emission ratio and the change inthe emission ratio upon coexistence with 8-Br-cGMP is shown in FIG. 5.

From this result, the responses of cGMP-expressing CHO-K1 cells toaddition of 8-Br-cGMP were confirmed, but it was revealed that thelinker sequences SEQ ID NO:NO. 1 (MDELKY) and SEQ ID NO: 2 (YPYDVPDYAN)both showed no significant effect on either the initial emission ratioor the emission ratio.

No significant difference in the initial emission ratio was observedbetween CGY-A12 and CGY-DEL 1 to 4; therefore, it is suggested thatintroducing various flexible domains into the polypeptide chain mayoptimize the structure of the cGMP-visualizing probe of the presentinvention.

These results indicate that CGY-Del 1 to 4 are preferable as thecGMP-visualizing probes for measuring intracellular cGMP levels.

Example 6 Introduction of CGY into Human Embryonic Kidney Cells

Human embryonic kidney cells (HEK293) were cultured in Dulbecco'smodified eagle medium supplemented with 10% FCS, 1 mM sodium pyruvate,and 0.1 mM nonessential amino acids at 37° C. in 5% CO₂, to give HEK293cells.

The resulting HEK293 cells were transfected by the same method as inExample 2 with CGY-Del1, CGY-Del2, CGY-Del3 and CGY-Del4 expressionvectors, to give HEK293 cells expressing each type of CGY.

Example 7 Quantification of cGMP in the Living Cells by thecGMP-Visualizing Probes

The cGMP-visualizing probes were used to detect intracellular cGMPgenerated upon stimulation of the living cells with nitrogen monoxide(NO).

First, to activate the soluble guanylyl cyclase in the HEK293 cellshaving CGY-Del1 introduced therein obtained in Example 6, NOC-7, acompound for releasing NO spontaneously in a rate-controlled manner, wasadded to give its final extracellular concentration of 500 μM.

FIG. 6 shows a photomicrograph of emissions induced by NOC-7 in theCGY-Del1-expressing HEK293 single cell.

FIG. 6 indicates that the emission ratio was rapidly decreased uponaddition of 500 μM NOC-7 to the CGY-Del1-expressing HEK293 cells, and asubsequent increase in the emission ratio to nearly the initial levelwas observed.

FIG. 7 shows changes in the NOC-7-induced emission ratio for theCGY-Del1-expressing HEK293 single cell.

The emission ratio was first rapidly decreased followed by a slowerincrease in the ratio.

Then, when the CGY-Del1-expressing HEK293 cells were treated with 100 μMzaprinast (selective inhibitor for cGMP-specific phosphodiesterase), thesame initial rapid decrease in the emission ratio was observed. However,the later slower increase in the emission ratio was not observed.

When the cells were pre-treated with 10 μM ODQ (selective inhibitor forNO-sensitive guanylyl cyclase), the change in the emission ratioelicited by NOC-7 was not observed.

From FIG. 7, it was suggested that the decrease in the emission ratiofollowed by an increase in the ratio is attributable to decomposition ofcGMP by cGMP-specific phosphodiesterase.

Similar results were also obtained by using the HEK293 cells expressingCGY-DEI 2 to 4.

From the above results, it was revealed that the occurrence of cGMP inliving cells could be confirmed by using the cGMP-visualizing probes ofthe invention of the present application.

Example 8 Measurement of Intracellular cGMP by the cGMP-VisualizingProbe

Accordingly, 1 mM 8-Br-cGMP, a phosphodiesterase-resistant analogue ofcGMP, was added to the system described above.

The results are shown in FIG. 8.

Following a decrease and a subsequent increase in the emission ratio,the ratio was decreased again by adding 1 mM 8-Br-cGMP. By usingzaprinast in place of 8-Br-cGMP, a similar re-increase was confirmed inthe system where the emission ratio was recovered.

From the foregoing results, it was confirmed that the response ofCGY-Del1 in detecting cGMP is reversible, and by using thecGMP-visualizing probe of the invention of the present application, thefluctuating concentration of cGMP in living cells can be measured.

INDUSTRIAL APPLICABILITY

As described in detail above, the present invention provides acGMP-visualizing probe that enables the easy detection andquantification of cGMP with high accurately even in vivo, as well as amethod of detecting and quantifying cGMP by using the same.

1. A method for detecting and quantifying cGMP, which comprises: (1)introducing a polynucleotide expressing a cGMP-visualizing probe intocells, whereby the cGMP-visualizing probe is expressed and made tocoexist with cGMP, wherein the cGMP-visualizing probe comprises: (a) apolypeptide that binds specifically to cGMP and is obtained by deletingamino acids 1 to 47 of cGMP-dependent protein kinase Iα (PKG Iα), and(b) two chromophores with different fluorescence wavelengths, one ofwhich is linked to the N-terminal and the other of which is linked tothe C-terminal of the polypeptide, wherein the chromophores are capableof exhibiting fluorescence resonance energy transfer (FRET); and (2)measuring the change in fluorescence wavelength.
 2. The method of claim1, wherein the chromophores are cyan fluorescent protein linked to theN-terminal of the polypeptide and yellow fluorescent protein linked tothe C-terminal of the polypeptide.
 3. The method of claim 1, wherein theprobe optionally comprises linker peptides between said chromophores andsaid polypeptide.